Patent Application
20200063189
The present disclosure relates generally to nucleic acid purification and amplification by the polymerase chain reaction (PCR). More particularly, the present disclosure relates to compositions, systems and methods for performing nucleic acid purification and amplification in a point of care system or device.
Point of care diagnostic systems and methods provide medical professionals with timely diagnostic information without the use of more expensive and time consuming lab-based tests. Typical point of care tests are performed at patient-provider contact, either in a clinician's office or clinic, in a field clinic or field trial, a provider home visit, or similar situation.
As such, many valuable diagnostic tests have been considered impractical for point of care diagnostics. For example, many nucleic acid amplification tests identify specific disease vector nucleic acids to accurately diagnose infections and their causes, but are not practical for point of care diagnostics. To amplify nucleic acids from biological specimens, the nucleic acids need to be purified in order to remove digestive enzymes, inhibitor molecules and other contaminants present in the specimen that would inhibit nucleic acid amplification reactions, such as RT-qPCR and PCR reactions. In addition, these tests are extremely sensitive to environmental contamination, an issue in many point of care settings both within and outside of medical facilities. In short, existing nucleic acid purification and amplification methods are time consuming, may use relatively large volumes of reagents, require separation or centrifugation steps, or utilize expensive microbead or similar substrates that require time consuming recovery and recycling. It would therefore be advantageous to provide devices, compositions and methods of separating, amplifying and qualitatively and/or quantitatively analyzing nucleic acids that could be used in a point of care device. The present disclosure is pertinent to this need.
There is a need in the art for nucleic acid extraction, purification and analysis methods and systems that can be implemented with low volumes of reagents and without using beads or centrifugation. This disclosure addresses the foregoing concerns by providing for the purification of nucleic acids in a microfluidic environment. While existing lab methods for nucleic acid purification using silica-based chromatography require reagents and methodology that are not suitable for use on a chip-based, stand-alone diagnostic device, the present disclosure is designed to facilitate extraction, separation, amplification and analysis of nucleic acids on the same device as the subsequent quantification assay.
In one aspect, the present disclosure provides a method for the purification of nucleic acids from a biological sample, including the steps of delivering the unpurified nucleic acids into a microfluidic region, contacting the nucleic acids with a fixed surface in the microfluidic region, wherein the nucleic acids adhere to the surface; washing the microfluidic region and surface with a first buffer; washing the microfluidic region and surface with a second buffer, where the second buffer has a pH that is equal to or higher than the first buffer.
In one aspect, the fixed surface comprises a silicon oxide or metal oxide or nitride. In one aspect, the metal oxide comprises, aluminum oxide, or hafnium oxide.
In one aspect, the present disclosure also provides for amplifying at least some of the nucleic acids to produce an amplification product; and detecting the amplification product. In one aspect, delivering the nucleic acids to the microfluidic region includes adding a biological sample to an adhesion buffer, disrupting cells, viruses or bacteria in the adhesion buffer so that the nucleic acids are mixed with the adhesion buffer. Disruption can include cell/virus lysis. The lysis can be performed using any suitable approaches, which include but are not necessarily limited to thermal, chemical and mechanical based lysis.
In one aspect, the present disclosure provides a method for the purification, amplification, and detection of the presence or absence of a nucleic acid in a sample. In embodiments the disclosure provides for generation and analysis of nucleic acid amplification products, including the steps of delivering the unpurified nucleic acids into a microfluidic region, contacting the nucleic acids with a fixed surface in the microfluidic region, wherein the nucleic acids adhere to the surface; washing the microfluidic region and surface with a first buffer; washing the microfluidic region and surface with a second buffer, where in certain approaches the second buffer has a pH that is equal to or higher than the first buffer; amplifying at least some of the nucleic acids to produce an amplification product(s); and detecting the amplification product(s). In one aspect, delivering the nucleic acids to the microfluidic region includes obtaining a biological sample known to or suspected of comprising or potentially comprising nucleic acids, adding the sample to an adhesion buffer, and lysing or otherwise disrupting cells and/or viruses in the adhesion buffer so that the viral or cellular contents are mixed with the adhesion buffer. In one aspect, the step of delivering the nucleic acids to the microfluidic region uses an adhesion solution comprising kosmotropic salts and a nuclease inhibitor. In embodiments, one or more solutions, including but not limited to buffers, used in embodiments of the disclosure are free of organic solvents. In embodiments the solutions are ethanol free, are chaotropic salt-free, or are both organic solvent and chaotropic salt free. Those skilled in the art will recognize that in certain cases the term “free” may nevertheless comprise trace amounts of chaotropic salts, or organic solvents which include but are not necessarily limited to ethanol or other alcohols that will be apparent to those skilled in the art given the benefit of the present disclosure.
In one aspect of the present disclosure, the purification and amplification steps take place in or on the same device. In one aspect, purification, amplification, and detection all occur in the same device. In one aspect, following detection of the amplification products, the method includes determining the identity of the source of the nucleic acids and reporting this result to the diagnostic provider, creating a record of this result in computer readable media, or both.
The accompanying drawings provide visual representations that will be used to more fully describe the representative embodiments disclosed herein and can be used by those skilled in the art to better understand them and their inherent advantages.
Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
The present disclosure related generally to compositions, methods and devices for nucleic acid analysis. Embodiments comprise separating nucleic acids from cellular and/or viral non-non-nucleic acid components, and detecting and/or quantifying the nucleic acids.
In more detail, clinical laboratory-based nucleic acid amplification tests (NAT) play an important role in diagnosing viral infections but their laboratory infrastructure requirements and failure to diagnose at the point of need limit their clinical utility in both resource-rich and—limited clinical settings. The development of fast and sensitive point-of-care (POC) viral NAT may overcome these limitations. The scalability of silicon microchip manufacturing combined with advances in silicon microfluidics present an opportunity for development of rapid and sensitive POC NAT on silicon microchips.
Methods of the present disclosure are implemented using devices comprising chips, which may comprise channels, and wherein on-chip nucleic acid separation, amplification and detection/quantification is performed. The disclosure comprises each process step and all combinations of process steps described here, each component and all combinations of devices and the devices themselves as described herein, each reagent and combinations of reagents described herein, and all combinations of the foregoing components. The methods disclosed herein minimize or entirely avoid the use of the organic solvents and other chaotropic agents that are commonly used in nucleic acid extraction. Thus, certain implementations of this disclosure have advantages in that they avoid use of components that are unsuitable for use outside of clinical or laboratory settings, would increase cost of manufacture, and would otherwise require special shipping/handling of a finished point of care device.
Components pertinent to implementing embodiments of this disclosure comprise one or more fixed surfaces (e.g. silicon wafer, silicon pillars), which may be coated with certain compositions that include but are not limited to a silicon oxide, or a metal oxide, a binding/lysing buffer, one or more wash buffers, one or more elution buffers, the latter of which may also function as amplification buffers.
The surface(s) are present or contained within a microfluidic environment having a volume in certain embodiments from 10 microliters to 1500 nanoliters. In embodiments the volume is less than 5 microliters, less than 2 microliters, or between about 500 to 1500 nanoliters. It is considered without being bound to any particular theory that such reaction volumes and surface areas allow for precise control of purification and amplification, for example, by rapid and precise temperature control, and rapid and precise changes in the microfluidic or nanofluidic shell surrounding each individual nucleic acid to be purified and amplified.
The surfaces are in embodiments silicon oxides or metal oxides or nitrides, such as aluminum oxide (Al2O3), hafnium oxide (HfO2), silicon nitride (Si3N4), or silicon oxide (SiO2). The surfaces provide for adherence (e.g., non-covalent) of the nucleic acids, and retention of the nucleic acids at certain temperatures or in certain microfluidic solutions, and efficient release of the nucleic acids at other temperatures or in other microfluidic solutions. The surfaces are made by techniques such as chemical vapor deposition (CVD) techniques on/within microfluidic vessels produced in part using, for example, silicon oxide wafers. A surface where nucleic acid binding, and/or amplification, and/or detection is performed, may be either open (i.e., flat or smooth) space or it may comprise three dimensional features that are within or on a surface, such as pillars, which can improve surface to volume ratio.
In embodiments three dimensional features of a microfluidic vessel, or a portion thereof, or a surface in a microfluidic vessel of this disclosure, are formed using any suitable approach by modifying a substrate, such as a silicon wafer or silicon nitride substrate. In embodiments the substrate is modified at least in part by a process that comprises etching. By “etching” it is meant that layers are removed from a surface of substrate, such as a wafer, during manufacturing. Given the benefit of this disclosure those skilled in the art will be able to adapt any suitable etching or other approaches to produce surfaces that can be used in various embodiments of the present disclosure, as further described below. In certain approaches etching comprises laser etching, liquid phase etching or plasma phase etching. Liquid phase etching can comprise wet etching or anisotropic wet etching. Similarly, plasma etching can be isotropic or anisotropic. In embodiments a silicon wafer is modified for use in various implementations of this disclosure by deep reactive ion etching. Devices, reagents and methods for deep reactive ion etching are known in the art and can be adapted by those skilled in the art, given the benefit of the present disclosure, to produce microfluidic devices and/or components thereof having surface areas that comprise three-dimensional features, including but not necessarily limited to micropillars.
Micropillars comprise dimensions that are suitable for use in the methods and microfluidic components/devices of this disclosure. In non-limiting examples, the micropillars are columnar, and thus may be rectangular or they may have a rounded shape. In embodiments the micropillars are from 190-200 μm in length. The length may be perpendicular relative to a substrate from which the micropillars project, with the understanding that the micropillars may be formed of the same material as the substrate. In certain embodiments the micropillars have a width or diameter of approximately 20 μm. The micropillars are generally configured on a surface such that they provide for adequate flow-through dynamics and surface area whereby a biological sample comprising nucleic acids can be contacted with the micropillar surface area such that at least some of the nucleic acids adhere to the micropillar surface, as further described herein. In certain implementations the micropillars are present in a vessel component of a microfluidic device, such as a chip, and are spaced such that they have a center-to-center distance of about 50 μm. In embodiments interpillar distance is about 30 μm. A representative and non-limiting image of a scanning electron micrograph of a cross section of a chip comprising micropillars is presented in
After micropillars are formed, regardless of the particular technique(s) used in their formation, the micropillars are coated with a suitable material, such as the silicon oxides or metal oxides as described herein. In certain embodiments the micropillars are coated with, for example, Al2O3, HfO2. Si3N4, or SiO2, using any suitable approach. In one approach chemical vapor deposition (CVD) is used. CVD techniques are known in the art and given the benefit of the present disclosure can be adapted for use in embodiments of this disclosure to, for example, coat micropillars with Al2O3, HfO2, Si3N4. In certain approaches the micropillars can be coated with SiO2 using approaches that are distinct from CVD, such as by thermal oxidation of silicon. Those skilled in the art will readily be able to adapt well known parameters of oxide growth kinetics during thermal oxidation of silicon to achieve suitable SiO2 micropillar coating. In embodiments, micropillars of this disclosure comprise an outer layer of Al2O3, HfO2, Si3N4, or SiO2 that has a thickness of 10-500 nm, inclusive, and including all numbers and ranges of numbers there between.
As discussed above, the micropillars may be present in a vessel. Any suitable vessel can be employed such that samples comprising nucleic acids as described herein can be isolated, and/or purified, and/or analyzed. Generally the vessel has any shape that permits fluid sample flow, including but not necessarily limited to a straight vessel, a vessel having bends comprising corners, or curved bends. In embodiments the vessel has a serpentine shape, and thus has one or more bends or meanders. In non-limiting embodiments a serpentine vessel has from 4-12 bends. The vessel has a total fluid volume capacity that can vary depending on the particular implementation and by changing the area of its footprint and/or its depth. In general a suitable vessel has a fluid capacity volume of not less than 1 μL. In non-limiting examples the vessel fits into an area of from 4.0 mm×6.0 mm, inclusive and including all numbers and ranges of numbers there between. In an embodiment the disclosure includes a vessel comprising a pillar chamber that is approximately 4.5 mm×5.0 mm. As one non-limiting illustration, a schematic of a vessel design is shown in
In certain examples used herein, micropillars formed as described above are scraped from the wafer in which they are formed. The scraped micropillars are then used in solution to perform a nucleic acid assay that is designed to mimic an internal vessel environment in terms of volume, buffer components, amplification and detection reagents, time, temperature, micropillar density, surface area, pH, etc.
In various embodiments the disclosure includes any surface(s) described herein, wherein the surfaces are in non-covalent association with nucleic acids. In embodiments the disclosure comprises a plurality of micropillars coated with a composition comprising or consisting essentially of Al2O3, HfO2, Si3N4, or SiO2 as described herein, wherein the polynucleotides are in a non-covalent physical association with the surface of the micropillars, i.e., the polynucleotides are in a complex with the micropillars. Those skilled in the art will recognize that the physical associations/complexes as described herein may be transient and are subject to thermodynamic, fluid dynamic, biochemical factors, buffering conditions, equilibriums, etc., that may be present during the performance of any nucleic acid isolation, detection, quantitation or quantification process of this disclosure.
The solutions, also referred to herein as buffers, used in the present disclosure include one or more of each of: a lysing buffer, a binding buffer, a wash buffer, and an elution buffer. In certain embodiments, the binding/lysing solution has an acidic buffered pH (e.g., 0, 1, 2, 3, 4, 5, 6, or 7), includes one or more salts, including kosmotropic salts and NaCl, and optionally a nuclease inhibitor and/or a proteinase. In certain embodiments, this solution has a pH of between about 1 and 5. For example, the solution may have a pH of about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0. In certain embodiments, the solution has a pH from about 2.0 to 4.0. In one embodiment, the solution has a pH from about 2.5 to 3.9. In certain embodiments, the salt constituents include NaCl from about 0.5M to 2.0M (e.g., 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, and 2.0M), and kosmotropic salts from about 0.01M to 5M. For example, the kosmotropic salts may have a concentration of about 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.11M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, 0.19M, 0.2M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0.29M, 0.3M, 0.31M, 0.32M, 0.33M, 0.34M, 0.35M, 0.36M, 0.37M, 0.38M, 0.39M, 0.4M, 0.41M, 0.42M, 0.43M, 0.44M, 0.45M, 0.46M, 0.47M, 0.48M, 0.49M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, and 5.0M. In certain embodiments, the kosmotropic salts have a concentration of about 0.1M to 3M. In one embodiment, the kosmotropic salts have a concentration of about 0.1 to 1.0 M. In one embodiment, the kosmotropic salts have a concentration of about 0.1M to 0.35M. The nuclease inhibitor constituent prevents or reduces the contamination of the purified nucleic acids by ribonucleases that could degrade the purified nucleic acid, and include, for example PROTECTOR RNase Inhibitor (Roche), SUPERaseIn™ (ThermoFisher Scientific), RNaseOUT™ (ThermoFisher Scientific), RNase Inhibitor (ThermoFisher Scientific) and RNasin™ (Promega). In certain embodiments, the concentrations of inhibitor are from about 1-2 U/μL. For example, the concentration of the inhibitor may be about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 U/μL. The proteinase constituent denatures and degrades protein contaminants and proteins released through lysis that would interfere with nucleic acid amplification, and include, for example, Proteinase K (Geriaid), also known as Peptidase K or Endopeptidase. In certain embodiments, the concentrations of the proteinase are from about 0.001-100 U/μl. In one embodiment, the concentration of the proteinase is from about 0.01-10 U/μl. Kosmotropic salts provide cations or anions that contribute to the ordered stability of a polar solvent (e.g., water). In certain embodiments, the kosmotropic salts are included in some solutions or buffered solutions. The kosmotropic salts may be, for example, sulfates, acetates, carbonates, and phosphates, such as (NH4)2SO4 (ammonium sulfate), CH3COONa (sodium acetate), H2CO3 (carbonic acid and its basic species), and K2HPO4 (potassium phosphate and its acidic/basic species). In embodiments the methods disclosed herein minimize or entirely avoid the use of the organic solvents and other chaotropic agents that are commonly used in nucleic acid purification. Without intending to be bound by any particular theory it is considered that these components are not suitable for use outside of clinical or laboratory settings, would increase cost of manufacture, and would otherwise require special shipping/handling of a finished point of care device.
In some embodiments, the one or more wash solutions include at least a first wash buffer and a second wash buffer. Wash buffers function to remove lysing debris and other contaminants from the nucleic acids after the nucleic acids have adhered to the surface. In certain embodiments, the first wash buffer is mildly acidic and contains kosmotropic salts, a reducing agent and may include, for example, an RNase inhibitor. In one embodiment, the first wash buffer has a pH below 7. For example, the first wash buffer may have a pH of about 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9. In certain embodiments, the first wash buffer has a pH below about 5. In one embodiment, the first wash buffer has a pH below about 4. Kosmotropic salts may be selected as described above, and may be present in a concentration of from 0, 1M to 5 M. For example, the kosmotropic salt may have a concentration of from about 0.1M, 0.11 M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, 0.19M, 0.2M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0.29M, 0.3M, 0.31M, 0.32M, 0.33M, 0.34M, 0.35M, 0.36M, 0.37M, 0.38M, 0.39M, 0.4M, 0.41M, 0.42M, 0.43M, 0.44M, 0.45M, 0.46M, 0.47M, 0.48M, 0.49M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0 M, 1.M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4.0M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, to about 5.0M. In certain embodiments, the kosmotropic salt has a concentration of from about 0.2 to 4.0 M. In some embodiments, the kosmotropic salt has a concentration of from about 0.5 to 2.5M. Reducing agents of the present disclosure include, but are not limited to DTT (dithiothreitol) and TCEP (tris(2-carboxyethyl)phosphine). The reducing agent may be present at a concentration from about 0.1 mM to about 20 mM. For example, the reducing may be present at a concentration from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 mM. In some embodiments, the reducing is present at a concentration from about 1 to 10 mM. In some embodiments, the reducing is present at a concentration from about 1 to 10 mM, with an RNase inhibitor as selected and described above.
Additional wash buffers may differ in pH and other properties, for example, possess lower or higher ionic strength, lower or high pH, or lower or higher concentrations of kosmotropic salts or RNase inhibitor, with adjustments in composition and concentration of solutes made to improve the purification of the adhered nucleic acids without reducing their adherence to the surfaces. A second wash buffer with lower ionic strength may be used to remove additional contaminants or residual salts remaining from the first wash buffer. In certain embodiments, the second wash buffer is mildly acidic, contains kosmotropic salts, and optionally, lower amounts of reducing agent and lower amounts of RNase inhibitor, if any. In some embodiments, the second wash buffer has a pH below 7. In one embodiment, the second wash buffer has a pH below pH 5. In some embodiments, the second wash buffer has a pH of about 3.5 to 4.5. Kosmotropic salts may be selected as described above, and may be present in a concentration of from 0.1 to 50 mM. For example, the kosmotropic salt may have a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mM. In some embodiments, the kosmotropic salt has a concentration of about 1 to 40 mM. In some embodiments, the kosmotropic salt has a concentration of about 10 to 25 mM. For example, a second wash buffer that has a pH of about 4.0 buffered with 10 to 25 mM ok a kosmotropic salt (e.g., NaOAc) may be employed.
The elution buffer (which may also function as the amplification buffer) dislodges the nucleic acids from the surface. For methods where amplification is performed while the nucleic acids are adhered, the elution buffer is administered following amplification. Where amplification is performed in solution, the elution buffer is used to dislodge the nucleic acids, with the amplification reagents added at the same time or thereafter. In certain embodiments, amplification and purification are performed in the same microfluidic space or reactor. In certain embodiments, the elution buffer has a low ionic strength and a neutral to slightly basic pH. For example, an elution buffer may have a pH from 6 to 10. For example, the elution buffer has a pH of 6, 7, 8, 9, or 10. In some embodiments, the elution buffer as a pH of about 8.5. In one embodiment, the elution buffer as a pH of about 8.5, contains about 10 to 25 mM Tris (Tris(hydroxymethyl)aminomethane), and from about 0% to 35% DMSO (dimethylsulfoxide). Without being bound by any particular theory, 0% to 35% refers to v/v. In some embodiments, the elution buffer contains about 5 to 25% DMSO, as a stabilizer. Thus, the elution buffer/amplification buffer in certain embodiments may be free of DMSO or may contain only trace amounts of DMSO. In some embodiments, the disclosure comprises lysing viral and/or cellular components and binding nucleic acids to a surface in a single step, and the same solution is thus used in the binding step and lysing steps, as this increases the speed of the overall purification, amplification and detection of the nucleic acids.
In some embodiments, the disclosure facilitates detection of threshold amounts of nucleic acids in the sample that is tested. As a non-limiting and hypothetical example, if a sample comprises 100 copies of a viral genome, in embodiments the disclosure is suitable for detecting at least 1 of the copies of the genome, and can comprise detecting from 4-100 of the copies, inclusive, and including all numbers and ranges of numbers there between. In some embodiments, the disclosure comprises generating a positive result with as few as four copies of a nucleic acid, such as an HCV genome. In some embodiments, from 1×107 to 4×107 copies are detected.
While non-limiting demonstrations of this disclosure are provided using plasma comprising known RNA inputs, it is expected that any biological sample, or other sample that comprises, or is or could be suspected to comprise, or is known to comprise nucleic acids, can be used in embodiments. In some implementation the sample comprises environmental samples, such as samples of water, food substances, or samples taken from an inanimate object, or an inanimate surface, including but not limited to devices or other flat or three-dimensional objects, devices, etc. In embodiments the sample is a biological sample. The biological or other sample may be used directly or it may be subjected to a processing step before being applied to a device of this disclosure. In embodiments the biological sample comprises a liquid biological sample, including but not limited to blood, plasma, urine, cerebrospinal fluid, lymph, saliva, sweat, semen, and lacrimal secretions. The biological sample may be a processed solid biological sample, such as a biopsy that has been subject to a mechanical disaggregation and/or may be subjected to one or more solutions. The biological sample may be obtained from a human or a non-human mammal or an avian animal using any suitable technique. Thus, in certain aspects the disclosure is pertinent to diagnostic applications in the field of veterinary medicine, in addition to human medical applications.
The polynucleotides isolated/amplified/detected/quantitated/quantified using approaches of this disclosure are not particularly limited. In general the polynucleotides will be of adequate length such that they are subject to amplification, including but not necessarily limited to amplification by methods that involve a polymerase chain reaction (PCR), including but not limited to Real-Time PCR (RT-PCR), i.e., quantitative RT-PCR (qPCR), as described further herein. The polynucleotides may be single or double stranded, or partially single or double stranded, and may be RNA or DNA. In some embodiments, the polynucleotides are RNA molecules, and their amplification can include a reverse-transcriptase for cDNA generation and further amplification and/or quantification. The type and/or origin of the nucleic acid that is determined using embodiments of this disclosure is not particularly limited and can come from, for example, any microorganism, which include but are not necessarily limited to pathogenic microorganisms. The microorganisms may be prokaryotic or eukaryotic. “Microorganisms” for the purposes of this disclosure also comprise viruses. In embodiments the microorganisms are selected from fungi, bacteria, archaca, viruses, and protozoans, including parasitic protozoans. In embodiments the disclosure relates to identifying nucleic acids from pathogenic bacteria. It is considered the disclosure can be used with any genus, species, or strain of bacteria. In certain examples the disclosure is used to detect nucleic acids from intracellular parasites.
With respect to viruses, while it is expected that any virus can be analyzed, in certain embodiments the virus is characterized by a single stranded RNA genome. The genome may be a (+) strand or a (−) strand. The viruses may be enveloped or non-enveloped. In embodiments the viral RNA is a viral genome from the viral family Filoviridae, or Paramyxoviridae, or Rhabdoviridae, or Bunyaviridae, or Arenaviridae, or Orthomxroviridae (including all types of influenza viruses). In embodiments the viral RNA is a viral genome or fragment thereof from the viral family Picornaviridae, Astroviridae, Caliciviridae, Hepeviridae, Flrvivindae, Togavindae, Arteriviridae, and Coronaviridae. In specific embodiments the viral RNA is from a human immunodeficiency virus (HIV), a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a cytomegalovirus, a human lymphotropic virus, an Epstein-Barr virus, a parvovirus, a paramyxovirus, or a herpes simplex virus. In other embodiments the RNA is an mRNA, or is a non-coding RNA. In embodiments the RNA comprises a snoRNA, an miRNA, or a mitochondrial RNA. In certain embodiments the RNA may be initially present in the biological sample as a component of a membranous vesicle, including but not limited to an exosome. In embodiments the RNA may be a circulating RNA of any type that is indicative of a disorder, including but not necessarily limited to cancer. Non-limiting embodiments of this disclosure are illustrated using HCV, HIV, Zika, HPV-16, and HPV-18.
In certain non-limiting embodiments a biological sample comprising, or suspected of comprising, a polynucleotide is subjected to a composition or process that is intended to disrupt the cell, virus or other substance in which the polynucleotides to be analyzed may be present. In certain embodiments, disruption comprises lysis of a cell or disrupting cell membranes, and/or disrupting a viral particle such that nucleic acids within the cell or the viral particle become amenable to binding to a surface of this disclosure. In non-limiting embodiments the sample is subjected to a chemical treatment, a thermal treatment, or a mechanical treatment (including but not limited to sonication) or a combination thereof, such that nucleic acids in the samples if present become, or are prepared to become, accessible to surfaces of this disclosure. In certain embodiments, lysis is performed using a chemical treatment that may include, for example, any of a variety of components which include but are not limited to detergents. Suitable detergents are known in the art and include for example SDS, which can be used at any suitable concentration, such as 1%, and NP-40, which can be used at for example, 0.5%. In certain approaches the sample can be subjected to a processing step in a device component, such as a cartridge, wherein the sample is exposed to any one or combination of the aforementioned compositions and/or conditions. The sample may also be subjected to, for example, a mechanical pressure that causes a fluid component of the sample to pass through a separation material such as a membrane having any suitable degree of porosity. The mechanical pressure may be adequate to pass some or all of the sample volume into and/or partially or fully through a microfluidic vessel described herein. In embodiments, the sample travels through the device without mechanical pressure and instead migrates via capillary action. In embodiments a wicking material can be included. In embodiments the sample can be subjected to heat that is provided by any suitable source or apparatus, for example, by an on-board exothermic chemical reaction component. The same approach can be adapted for heating that occurs, for example, during amplification reactions, and the process may further employ endothermic chemical reaction components for cooling purposes—thus, in certain embodiments a device of this disclosure can operate independent of batteries or other sources of electric power, further providing advantages for point of care applications in a wide variety of settings, including but not necessarily limited to the scene of a medical emergency, including but not limited to a battle-field environment.
In certain embodiments a result obtained from using a method and/or device and/or system of this disclosure can be compared to any suitable reference, examples of which include but are not limited control sample(s), a standardized curve(s), and/or experimentally designed controls such as a known input polynucleotide value used to normalize experimental data for qualitative or quantitative determination of the amount of polynucleotide, or a cutoff value, such controls being useful if desired to normalize for mass, molarity, concentration and the like. A reference value may also be depicted as an area on a graph. In embodiments the disclosure provides for an internal control that can be used to normalize a result, such as a signal that indicates an amount of nucleic acids. In embodiments the disclosure provides for use of calibrators, i.e., known inputs which can be used to test, establish, confirm, etc. the accuracy of any particular signal. In certain embodiments one or more calibrator and/or internal control samples can be stored on-chip, or they can be stored in a separate device component, including but not necessarily limited to a permanently fixed and/or detachable cartridge component. In certain aspects the disclosure includes calibrating each chip, or calibrating only selected chips from a group (i.e., a lot) of chips. In certain non-limiting examples, the disclosure includes a positive control and/or a calibrator in a distinct channel or other segment of a chip. In one implementation a lysis/binding buffer can pass over a segment where a known concentration of control RNA, which may comprise so-called armored RNA (comprising for example a complex of bacteriophage protein and RNA) is solubilized in the lysis/binding buffer and delivered to an extraction/amplification chamber. The armored RNA can be lysed, bound to pillars as described herein, washed and amplified/detected in the same way as the test sample. In embodiments a Ct value of the control will be used for comparison to a pre-determined standard curve that is associated with a specific lot of chips, and thus the standard curve can be adjusted based on the Ct value of the control, such as if it falls within a pre-defined range. Additionally or alternatively, embodiments of the disclosure can include an internal control, such as any suitable in vitro transcribed RNA if the sample is being tested for RNA, that can be used to assess assay parameters, performance, etc. In a non-limiting embodiment the internal control RNA comprises in vitro transcribed moss gene RNA. Such an internal control RNA will be subjected to analysis in both test and positive control channels and thus will be extracted, amplified and detected at the same time as test RNA, which can be performed in a multiplexed format. The internal control probe may be conjugated with a fluorophore or other detectable label that is spectrally distinct from the test probe fluorophore. Accordingly, increased fluorescence from both probes can be monitored concurrently and configured such that the internal control must fall within a pre-defined range for the test result to be considered valid.
In certain embodiments a result based on a determination of the presence, absence, or amount of a polynucleotide using an approach of this disclosure is obtained and is fixed in a tangible medium of expression, such as a digital file, and/or is saved on a portable memory device, or on a hard drive, or is communicated to a web-based or cloud-based storage system. The determination can be communicated to a health care provider for diagnosing or aiding in a diagnosis, such as of a bacterial or viral infection, or for monitoring or modifying a therapeutic or prophylactic approach for any disease, disorder or condition that is associated with the presence and/or amount of the polynucleotide in the sample.
In certain examples the disclosure comprises an article of manufacture, which in embodiments can also be considered kits. The article of manufacture comprises at least one component for use in the nucleic acid analysis approaches described herein and packaging. The packaging can contain a device and/or a chip comprising microfluidic vessels described herein. In various embodiments, the article of manufacture includes printed material. The printed material can be part of the packaging, or it can be provided on a label, or as paper insert or other written material included with the packaging. The printed material provides information on the contents of the package, and instructs user how to use the package contents for nucleic acid analysis. In embodiments the article of manufacture can comprise one or more suitable sealed, sterile containers that contain for example, buffers described herein or stock solutions, primers that are directed to known polynuclcotide sequences for any particular organism, primers for use in RT-PCR reactions, labeled probes for use in such reactions, enzymes, such as reverse transcriptase and a suitable DNA polymerase, RNAse inhibitors, nucleotides, etc. In one approach the package comprises a cartridge comprising one or more buffers used in nucleic acid extraction and/or for annealing nucleic acids to a surface in a device component.
In one example of the present disclosure, buffer, blood plasma or other biological specimen containing nucleic acids is added to the binding buffer, the mixture is heated to about 50 to 70° C. and incubated with a metal oxide- or silicon oxide-coated surface as described above to allow for nucleic acid binding. Taking advantage of the micro/nano fluidic scale, incubation times are, in certain embodiments, no more than 60 minutes. In some embodiments, incubation times are no more than 45 minutes. In some embodiments, incubation times are no more than 30 minutes. In some embodiments, incubation times are no more than 20 minutes. In some embodiments, incubation times are no more than 15 minutes. In some embodiments, incubation times are between about 1 and 15 minutes. For example, the incubation times are between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 minutes. Following this incubation, the surface is washed with a first wash buffer in order to remove contaminants. Next, the surface is washed with a second wash buffer to remove residual wash salts. Finally, the nucleic acid may be eluted from the surface using an elution buffer (and incubation at 55° C.) or further steps (i.e. amplification) may be performed with the nucleic acids still adhered to the surface.
In certain embodiments the fluorescence emission can be measured in one or more segments of the reaction chamber, as illustrated in
In embodiments, one or more segments of the vessel can be connected to or in communication with a digital processor and/or a computer running software to interpret the position, amount, intensity, etc. of the fluorescent signal. A processor may also be included as a component of a device comprising the chip, wherein the processor runs software or implements an algorithm to interpret fluorescence or another optically detectable signal, and generate a machine and/or user readable output. In an embodiment, a chip component can be integrated or otherwise inserted into an adapter that comprises a detection device, such as a camera, which may also comprise a processor for fluorescence detection. In embodiments, a computer readable storage media can be component of a device of this disclosure, and can be used during or subsequent to performing any assay or one or more steps of any assay described herein. In embodiments the computer storage medium is a non-transitory medium, and thus can exclude signals, carrier waves, and other transitory signals.
Examples 1 and 2 describe RT-qPCR assays designed as a basis for advancing embodiments of the disclosure to on-chip applications. Examples 3 and 4 were performed using blanket silicon wafers coated with hafnium oxide or aluminum oxide, the blankets comprising a diced rectangular piece of silicon wafer containing wells formed by an attached gasket. The blanket is a model for the surface of a microreactor of this disclosure. RNA is bound to the blanket, washed, eluted and quantified in a benchtop RT-qPCR. Examples 5, 6 and 7 were performed using silicon nanopillars scraped from a microfluidic reactor, where the nucleic acids adhered to pillar structures formed as described above. The pillars are representative of pillar surface coatings in a commercial embodiment of a reactor and were less dependent upon diffusion and higher recoveries. Notably, results in Examples 6 and 7 were obtained using RNA bound to silicon oxide-coated pillars; the RNA was not eluted off the pillars before quantification, thus supporting on-chip implementations using nanopillar coated surfaces. Example 8 demonstrates extraction of RNA by capillary flow (
The following Examples are meant to illustrate but not limit embodiments of this disclosure. For Examples 1 and 2, an HCV (Hepatitis C Virus) RNA detection assay was performed with a sensitivity of 4 copies (cp) per reactions. Multiple nucleic acid amplification assays were tested across a standard concentration range of 4×100-4×106 cp/reaction. These standard concentrations were selected based on the range of HCV RNA plasma concentrations measured in 98% of HCV-infected patients and performance specifications of upstream steps, plasma separation and RNA extraction. [See, for example, Ticehurst, et al. J Clin Microbiol. 2007 August; 45(8): 2426-2433.] These specifications included a maximum blood sample of 20 μL, 50% recovery of plasma from blood sample (blood contains ˜50% plasma, on average), and 40% recovery of RNA during the extraction process. Table 1 illustrates, using representative high and low-end copy number values along the spectrum of observed patient HCV RNA concentrations, the number of HCV RNA copies in each step along the process taking into account these specifications.
Thus, these assays had sufficient dynamic range (4×100-4×106 cp/reaction) to detect 98% of all HCV RNA-positive specimens. Testing was performed using an in vitro transcribed, HCV 5′ UTR RNA standard, described below, diluted to 1×100-1×106 cp/μL. Ten replicates of each sample were analyzed during performance testing.
Two RT-qPCR assays were screened for pretesting prior to use on microfluidic purification, amplification and detection. Assay (A) Superscript III-Tfi (Example 1) and Assay (B) Superscript III-Amplitaq360 (Example 2). Both assays utilize two enzymes: 1) MMLV-based reverse transcriptase (Superscript 111) that has been genetically engineered for a longer half-life at higher reaction temperature and decreased RNascH activity and 2) DNA polymerase cloned from either Thermus filiformis (Tfi) or Thermus aquaticus (AmpTaq360) that has been genetically engineered for enhanced processivity and stability. Both the reverse transcription (RT) and qPCR reactions are performed in the same well on a 96-well plate instead of performing the RT reaction in one well and then transferring a portion of the reaction to a qPCR well in order to model an RT-qPCR assay performed in a single reaction chamber on silicon microchip. In this “one-tube” reaction, the temperature is maintained at an optimal temperature for generation of cDNA (RNA to single-stranded DNA) by the RT enzyme. The temperature is then increased to inactivate the RT enzyme and activate the DNA polymerase. The cDNA is then amplified using normal PCR temperature cycling—high temperature to melt DNA duplexes and lower temperature to elongate DNA from annealed oligonucleotide primers. The amplified DNA is quantified through the use of a fluorescently labelled oligonucleotide probe. The probe comprises a sequence that is complementary to the target sequence, a fluorophore conjugated to the 5′ end, and a quencher molecule conjugated to the 3′ end. During qPCR amplification, the inherent 5′-3′ exonuclease activity of the DNA polymerase results in digestion of the probe in a 5′-3′ direction, release of the fluorophore, and alleviation of quencher-mediated suppression of the 5′ fluorophore fluorescence. The fluorescence is measured at the end of each qPCR cycle and compared to values obtained from standards in order to quantify the concentration of RNA in the sample. For both assays, an in vitro transcribed RNA standard was used to test the performance of each assay across a standard range of 4×100-4×106 cp/reaction. The RNA standard was supplied from Amsbio and produced from a plasmid template that contained the complementary full-length sequence of the HCV 5′ UTR region obtained from a standard lab isolate. The HCV 5′ UTR is a highly conserved region of the HCV genome so use of this sequence from this isolate should be representative of the natural diversity of HCV sequences. The RNA is produced using standard in vitro transcription techniques and template DNA is digested using DNase. The resulting product contains <0.01% template DNA contamination. The DNA contamination level is acceptable because this amount of DNA will not significantly contribute to signal generation and the standard is DNA free at concentrations <1×104 cp/μL.
This Example provides a description of analysis using Superscript III-Tfi RT-qPCR.
Reverse transcriptase: MMLV-based Superscript III (SSIII) supplied by Invitrogen; DNA polymerase: Thermus filiformis polymerase (Tfi) supplied by Invitrogen. Final reaction reagent concentrations: 40 mM Hepes-KOH (pH 8.1), 15 mM KCl, 15 mM (NH4)2SO4, 2% glycerol, 200 nM dNTP, 200 nM forward primer (5′-CCCCTGTGAGGAACTACTGT-3′), 400 nM reverse primer (5′-GACCACTATGGCTCTCCCG-3′), 200 nM Atto633-conjugated probe (5′-Atto633-AGCCATGGCGTTAGTATGAGTGTCG-IAbRQSp-3′), 3.0 mM MgCl2, 0.2 mg/mL. BSA, 3 mM DTT, 50 U SSIII, and 1.7 U Tfi polymerase.
A 1.67× concentrated master mix was prepared and 6 μL of this master mix was added to 4 μL of RNA. For this experiment, standards were diluted in 10 mM Tris pH 7.5 containing ng/μL carrier RNA to final concentrations ranging from 1×100-1×106 copies/μL. Temperature cycling parameters on LIGHTCYCLER® 480 (Roche Life Sciences) instrument were 55° C. for 15 min, 95° C. for 3 min, 50 cycles of 1) 95° C. for 5 sec, 2) 60° C. for 30 sec. Fluorescence was only monitored during cycles 11-50.
Data analysis—Raw fluorescence values from the LIGHTCYCLER® 480 (Roche Life Sciences) instrument were used to fit amplification curves using the R qpcR package (freeware statistical analysis program). Ct values for each replicate were assigned by determining the 2nd derivative maximum value for a 5-parameter sigmoidal fit curve. Average Ct values for each standard were plotted against the Log 10 concentration for each standard and the data were fit with a linear regression line. The slope of the line was used to determine reaction efficiency (efficiency=10(−1/slope)−1) and the back-calculated concentrations of each standard replicate were used to estimate sample reproducibility (standard deviation) of each standard dilution.
For validation of this assay, 10 replicates of each standard (1×100-1×106 cp/μL) were analyzed on the LIGHTCYCLER® 480 (Roche Life Sciences) instrument using the method described above, amplification curves analyzed using the R qpcR package, standard curves generated, and assay performance was evaluated, as described above.
1Log 10 cp/μL,
2mean,
3reproducibility,
4number of replicates detected
This Example demonstrates an embodiment using Superscript III-Amplitaq360 RT-qPCR. Reverse transcriptase: Superscript III (SSIII) supplied by Invitrogen, DNA polymerase: Thermus aquaticus polymerase (AmpTaq360) supplied by Invitrogen Final reaction reagent concentrations: 50 mM Tris (pH 8.3), 75 mM KCl, 200 nM dNTP, 200 nM forward primer (5′-CCCCTGTGAGGAACTACTGT-3′), 400 nM reverse primer (5′-ACCACTATGGCTCTCCCG-3′), 200 nM Atto633-conjugated probe (5′-Atto633-AGCCATGGCGTTAGTATGAGTGTCG-IAbRQSp-3′), 2.5 mM MgCl2, 0.2 mg/mL BSA, 3 mM DTT, 50 U SSIII, and 1.25 U AmpliTaq360 polymerase.
A 1.67× concentrated master mix is prepared and 6 μL of this master mix is added to 4 μL of RNA standard diluted in 10 mM Tris pH 7.5 containing 10 ng/μL carrier RNA to final concentrations ranging from 1×100-1×106 copies/μL. Ten replicates of each standard were tested. Temperature cycling parameters on the LIGHTCYCLER® 480 (Roche Life Sciences) instrument were 55° C. for 15 min, 95° C. for 3 min, 50 cycles of a) 95° C. for 5 sec followed by b) 60° C. for 30 sec. Fluorescence was only monitored over the last 40 cycles.
Data analysis—Raw fluorescence values from the LIGHTCYCLER®; 480 (Roche Life Sciences) instrument were used to fit amplification curves using the R qPCR package (freeware statistical analysis program). Ct values for each replicate were assigned by determining the 2nd derivative maximum value for a 5-parameter sigmoidal fit line. Average Ct values for each standard were plotted against the Log 10 concentration for each standard and the data were fit with a linear regression line. The slope of the line was used to determine reaction efficiency (efficiency=10(−1/slope)−1) and the back-calculated concentrations of each standard replicate were used to estimate sample reproducibility (standard deviation) of each standard dilution.
Raw fluorescence values from the LIGHTCYCLER® 480 (Roche Life Sciences) instrument were used to fit amplification curves using the R qpcR package (freeware statistical analysis program). Ct values for each replicate were assigned by determining the 2nd derivative maximum value for a 5-parameter sigmoidal fit line. Average Ct values for each standard were plotted against the Log 10 concentration for each standard and the data were fit with a linear regression line. The slope of the line was used to determine reaction efficiency (efficiency=10(−1/slope)−1) and the back-calculated concentrations of each standard replicate were used to estimate sample reproducibility (standard deviation) of each standard dilution.
For validation of this assay, 10 replicates of each standard (1×100-1× 106 cp/μL) were analyzed on the LIGHTCYCLER® 480 (Roche Life Sciences) instrument using the method described above, amplification curves analyzed using the R qpcR package, standard curves generated, and assay performance was evaluated, as described above.
Ten replicates of each standard dilution were analyzed using the SSIII-AmpTaq360 RT-qPCR assay and the amplification curves for the test are depicted in
1Log 10 cp/μL,
2mean,
3reproducibility,
4number of replicates detected
This Example demonstrates use of a HfO2 coated surface for purification of RNA. In particular, this Example demonstrates use of a solution containing Hepatitis C RNA as described above added to a silicon oxide blank having a surface coating of hafnium oxide.
This Example demonstrates extraction and purification of RNA from buffer on an Al2O3 coated surface. A solution containing Hepatitis C RNA was prepared as described above and added to a silicon oxide blanket having a surface coating of aluminum oxide.
This Example demonstrates extraction and purification of RNA in plasma from micropillar structures with a binding buffer pH of from 2 to 4. In more detail, a solution containing 1×105 Hepatitis C RNA in plasma was prepared and added to reaction surfaces having pillars of silicon oxide, hafnium oxide and aluminum oxide. A pH 4 wash buffer having a range of kosmotropic salts (Si—none, Hf—K2HPO4, Al—NaOAc) was used, followed by a basic elution buffer at 55° C. (Si—pH 9.5, Hf—9.5, Al—8.5), with other conditions as described above. Note, the pillars were removed from the reaction surface then treated with the elution buffer, and unbound fractions purified from the elution buffer with a Qiagen viral RNA purification kit. Elution and pillar-bound RNA was analyzed by an SSIII-AT360 assay. FIG. 9 shows the percentage of recovery of Hepatitis C RNA from the pillars, elution, and pillars+elution buffer, showing the advantages of using kosmotropic salts and pillar structures to improve adhesion to the reactor surface and increased nucleic acid recovery. In
This Example demonstrates extraction and purification of RNA from buffer on a pillar surface. The above protocol from Example 5 was performed using pillars scraped from a silicon oxide coated chip with plasma standards having concentrations of Hepatitis C RNA ranging from ranging from 1×100-1×106 copies/μL, and using binding buffer at pH 2.5 and a wash buffer at pH 2.3 Purification and positive detection of RNA in plasma was achieved at concentrations ranging from 10 copies to 1×106 per μL. As shown in Table 4 below, average recoveries even for extremely low concentrations of RNA were at or near 50% and above, approaching the recoveries obtained by commercially available kits.
1Log 10 cp/μL,
2mean,
3standard deviation,
This Example demonstrates recovery of RNA in distinct reaction conditions.
Data summarized in
For
This Example demonstrates extraction and amplification of RNA by capillary flow, lysis of HCV particles as quantified by a device sold under the trade name LIGHTCYCLER®, 480 for analysis of RNA, and use of an internal RNA control to normalize results.
To obtain the data shown in
This Example expands the examples above, and demonstrates sensitive (4 copies/reaction) RT-qPCR and qPCR assays detecting HCV, HIV, Zika, HPV 16, and HPV 18 on a benchtop real-time PCR instrument.
In more detail, in this Example, we developed rapid and sensitive NAT for a number of RNA and DNA viruses on the same silicon microchip platform. We first developed sensitive (limit of detection (LOD), 4 copies/reaction) one-step RT-qPCR and qPCR assays detecting HCV. HIV, Zika. HPV 16, and HPV 18 on a benchtop real-time PCR instrument. The same novel silicon microchip design with an etched meandering microreactor, integrated aluminum heaters, thermal insulation trenches and microfluidic channels for delivery of reagents was used for all on-chip experiments in this Example following demonstration of precise and localized heating of the microreactor using melting temperature analysis. Following minimal optimization of reaction conditions, the bench-scale one-step RT-qPCR and qPCR assays were successfully transferred to 1.3 μL silicon microreactors with reaction times of 25 min and no effect on the LOD. Also, the reproducibility and reaction efficiencies of the bench scale and on-chip assays was similar. Taken together, these results demonstrate that rapid and sensitive detection of multiple viruses on the same silicon microchip platform is feasible. Further development of this technology, coupled with silicon microchip-based nucleic acid extraction solutions, could potentially shift viral nucleic acid detection and diagnosis from centralized clinical laboratories to the POC.
The following materials and methods were used to obtain the results described in this Example.
Oligonucleotides and Standards
All primers and hydrolysis probes were designed using PrimerBlast (NCBI) and publically-available sequences (Genmed, NCBI) and synthesized by IDT technologies (Table 1). In vitro transcribed (IVT) viral RNA was purchased (Amsbio, Mass.) and used as a standard for each RNA target (Table 2). For the DNA standards, linearized pHPV-16 plasmid DNA (clone 45113D, ATCC, Virginia) and pHPV-18 plasmid DNA (clone 45152D, ATCC, Virginia) were used as standards.
Bench Scale Assay Development of RNA Viruses
All bench scale RT-qPCR assays were developed using a LightCycler480 instrument (Roche, Switzerland) and IVT RNA for each target was used as a standard. Amplification was performed using 10 μl, reactions containing 50 mM TRIS pH 8.3, 75 mM KCl, 200 μM dNTP Mix (Invitrogen, California), 200 nM forward primer (IDT Technologies, Iowa), 400 nM reverse primer (IDT Technologies, Iowa), 200 nM hydrolysis probe (IDT Technologies, Iowa), 0.2 mg/mL BSA (Thermo Fisher, Massachusetts), 3 mM DTT (Thermo Fisher, Massachusetts), 50 units SuperScript III (Thermo Fisher, Massachusetts) and 1.25 units of AmpliTaq360 polymerase (Thermo Fisher, Massachusetts). The HCV and HIV assays contained 2.5 mM MgCl2 (Invitrogen, California) while the Zika assay contained 3 mM MgCl2. The HCV assay cycling conditions included a 15 minute RT step at 55° C., 3 minute initial denaturation step at 95° C., and 50 cycles of 10 second denaturation at 95° C. and 30 second amplification at 60° C. for a total assay time of 70 minutes. Both the HIV and Zika assays used cycling conditions that included a 5 minute RT step at 55° C., 3 minute initial denaturation step at 95° C., and 50 cycles of 5 second denaturation at 95° C. and 10 second amplification at 60° C. for a total assay time of 51 minutes. The standard concentrations used in experiments ranged from 4×106-4×100 copies per reaction.
Bench Scale Assay Development of DNA Viruses
Bench scale assays were developed for DNA viruses HPV 16 and HPV 18. Amplification of the HPV targets was performed using 10 μl reactions containing 50 mM TRIS pH 8.3, 75 mM KCl, 200 μM dNTP Mix (Invitrogen, California), 200 nM forward primer (IDT Technologies, Iowa), 400 nM reverse primer (IDT Technologies, Iowa), 200 nM hydrolysis probe (IDT Technologies, Iowa), 0.2 mg/mL BSA (Thermo Fisher, Massachusetts), 3 mM DTT (Thermo Fisher, Massachusetts), and 1.25 units of AmpliTaq360 polymerase (Thermo Fisher, Massachusetts). The standard concentrations used in experiments ranged from 4×106-4×100 copies per reaction. The HPV 16 assay contained 1.5 mM MgCl2 (Invitrogen, California) while the HPV 18 assay contained 4.5 mM MgCl2. The cycling conditions for the HPV 16 assay included a 3 minute initial denaturation step at 95° C., and 50 cycles of 10 second denaturation at 95° C. and 30 second amplification at 60° C. for a total assay time of 67 minutes. The HPV 18 assay utilized the same protocol but with 30 second amplification at 62° C. instead of 60° C.
Bench Scale Data Analysis
For bench scale assays, Ct values were determined using the Lightcycler 480 software. Although 50 cycles were performed for each assay, the reaction fluorescence was not monitored during the first ten cycles. Therefore, at the end of each run, 10 cycles were added to the Ct values calculated using the LightCycler480 software. Average Ct values for each standard were plotted against the Log 10 standard concentrations and linear regression line was determined. The slope of the line was used to determine the reaction efficiency (efficiency=(10(−1/slope)−1) and the back-calculated concentrations of each standard replicate were used to estimate sample reproducibility (standard deviation) of each standard dilution.
Chip Fabrication and Characterization
The PCR reactor was fabricated using silicon-glass technology. Details of the fabrication have been reported previously6 and are briefly summarized here (
Transfer of Bench Scale Assays to Silicon Microchip Reactor
The optimized bench scale assays for both RNA and DNA targets were then transferred to the silicon microreactor with some modifications. All reagent concentrations were the same as the bench scale assay except the AmpliTaq360 concentration was increased to 5 units per reaction for all targets on-chip. The same standards used to develop each bench scale assay were tested on-chip with a range of 4×100-4×105 copies per reaction. Reactions were loaded into the reaction chamber and amplified according to the following cycling conditions. The HCV assay included a 5 minute RT step at 55° C., 3 minute initial denaturation step at 95° C., and 40 cycles of 5 second denaturation at 95° C. and 10 second amplification at 60° C. for a total assay time of 24.8 minutes. The cycling conditions for both the HIV and Zika assays included a 2.5 minute RT step at 55° C., 1.5 minute initial denaturation step at 95° C., and 50 cycles of 5 second denaturation at 95° C. and 10 second amplification at 60° C. for a total assay time of 25 minutes. The HPV 16 and HPV 18 assays included a 1.5 minute initial denaturation step at 95° C. and 50 cycles of 5 second denaturation at 95° C. and 10 second amplification at either 60° C. (HPV 16) or 62° C. (HPV 18) for a total assay time of 22.5 minutes. Between runs, chips were cleaned by incubating the microreactor in 10% bleach at 95° C. for 5 min followed by one wash with water at 95° C. for 5 mins and then two additional room temperature water washes.
On-Chip Image Analysis
During the RT-qPCR on-chip program, images of the reaction chamber were captured using the inverted fluorescence microscope. The first image was captured at the start of the run, the second image was captured at the end of the RT step, and the remaining images were captured at the end of each amplification cycle. All images were then analyzed using ImageJ analysis software (NIH). The reaction chamber was divided into 9 sections with each section encompassing a meander (
On-Chip Ct Determination
Ct values were determined for each standard replicate using the qPCR package in the R Studio software (RStudio, Boston, Mass.) by calculating the 2nd derivative maximum for a 5-parameter sigmoidal fit line. Average Ct values for each standard were plotted against Log 10 standard concentrations and a linear regression line was determined. The slope of the line was used to determine reaction efficiency (efficiency=(10(−1/slope)−1) and the back-calculated concentrations of each standard replicate were used to estimate sample reproducibility (standard deviation) of each standard dilution.
The following results were obtained using the materials and methods of this Example as described above.
Bench Scale RNA Assays
In order to test the performance of each bench scale RNA assay, three independent experiments were performed in which IVT RNA standard dilution series (4×106-4×100 copies/reaction) were tested. For all targets, each standard concentration was detected in each independent experiment suggesting an assay sensitivity of at least 4 cp/reaction for each target. The mean Ct values calculated across the three independent experiments were plotted against the corresponding Log 10 RNA concentrations for each target. The overall efficiency of each assay, derived from the slope of the linear regression line, was 107.8% for HCV, 109.8% for HIV, and 98.8% for Zika. In order to assess assay variability, the Ct values of each standard replicate was used to determine a back-calculated RNA concentration from the average efficiency curve across the three replicates. The average variability of all RNA targets was less than 0.5 Log 10 (HCV, 0.05 Log 10; HIV, 0.35 Log 10; and Zika, 0.10 Log 10).
Bench Scale DNA Assays
The performance of each bench scale assay was assessed using three independent experiments were performed in which plasmid DNA standard dilution series (4×106-4×100 copies/reaction) were tested. For both HPV 16 and HPV 18, each standard concentration was detected in each independent experiment indicating an assay sensitivity of at least 4 cp/reaction for each target. The mean Ct values calculated across the three independent experiments were plotted in the same way as the RNA bench scale assays and the resulting reaction efficiencies for HPV 16 and HPV 18 were 108.9% and 100.9%, respectively. The average variability, based on back-calculated concentrations, of both DNA targets was less than 0.5 Log 10 (HPV 16, 0.16 Log 10; and HPV 18, 0.08 Log 10).
Characterization of the PCR Microchip
Prior to on-chip assay development, a series of tests were performed. After mounting the chip on the PCB, the RTDs were calibrated in an oven to ensure correct measurement of the temperature during PCR. The temperature values and uniformity were further verified by melting curve analysis using DNA fragments with known melting temperatures close to the annealing temperature of the PCR primers (Tm=60° C. and 70° C.) and to the denaturation temperature (Tm=80° C.). At 80° C., temperature uniformity across the microreactor was 0.7° C. and the measured temperature was correct within 0.5° C. The ramp time from 60° C. to 95° C. was 2 s at a heater current of 0.1 A. The cooling time from 95° C. to 60° C. was 4 s and is fixed by the thermal design. During temperature cycling, the bulk temperature of the chip surrounding the PCR microreactor did not exceed 50° C., due to the thermal insulation trenches (
On-Chip RNA Assays
In order to assess the performance of the on-chip RNA assays, three independent experiments were performed during which each IVT RNA standard across the standard range (4×105-4×100 copies/reaction) was tested on a microchip. All standard replicates of each target were detected, suggesting an assay sensitivity of at least 4 cp/reaction. The mean Ct values calculated across the three replicates were plotted in the same way as the bench scale assays resulting in reaction efficiencies of 100.6%, 95.7%, and 103.9% for HCV, HIV, and Zika respectively. The average variability, based on back-calculated concentrations, across all standard concentrations of HCV, HIV, and Zika RNA were 0.35 Log 10, 0.41 Log 10, and 0.32 Log 10, respectively.
On-Chip DNA Assays
In order to assess the performance of the on-chip DNA assays, three independent experiments were performed during which each plasmid DNA standard across the standard range (4×105-4×100 copies/reaction) was tested on a microchip. All standard replicates of each target were detected, indicating an assay sensitivity of at least 4 cp/reaction. The efficiencies for HPV 16 and HPV 18 were 97.7% and 98.2% respectively. The average reproducibility, based on back-calculated concentrations, across all standard concentrations was 0.18 Log 10 for HPV 16 and 0.17 Log 10 for HPV 18.
It will be recognized that the foregoing results support the feasibility of detecting viral nucleic acids using novel RT-qPCR and qPCR assays and microreactors on silicon microchip. Bench scale RT-qPCR assays for HCV, HIV, and ZIKV and qPCR assays for HPV 16 and HPV 18 with assay sensitivities of 4 cp/reaction were developed using a standard bench scale real-time PCR instrument. PCR silicon microchips with 1.3 μL microreactors and rapid and consistent temperature cycling were designed and manufactured. Finally, these assays were successfully transferred to the developed PCR silicon microchips with little optimization and no loss in sensitivity (4 cp/reaction) and reproducibility (<0.5 Log 10). These results demonstrate that rapid and sensitive amplification of viral nucleic acids in a 1.3 μL microreactor is feasible and supports nucleic acid-based diagnostic devices using scalable silicon microchip technology.
Previously available POC serologic tests for diagnosis of acute viral infection suffer from decreased clinical sensitivity since delayed antibody production limits the sensitivity of immunoassays in the first week of illness. Also, once detectable, persistence of antibodies complicates the identification of current versus past infection. While diagnostic NAT are considered the gold standard for detection of viral infections, they require large, expensive equipment and skilled operators. As a result, prior to the present disclosure, viral NAT must be performed in centralized clinical laboratories which leads to a long time-to-result. All of these can increase diagnostic uncertainty at the POC leading to inappropriate antimicrobial use, a significant public health concern. Providing not only sensitive, but fast, POC viral NAT may overcome these limitations and enable clinicians to more accurately diagnose viral infections at the point-of-need. In this regard, the presently demonstrated five on-chip assays provide reproducible and sensitive detection of HCV. HIV, Zika. HPV-16, and HPV-18. Additionally, the silicon microchip technology utilized in this study allows for rapid and accurate thermal cycling and a shortened time-to-result. Indeed, our viral nucleic acid assays were performed in 25 minutes, significantly shorter than the 51 minute bench scale assay. Further optimization of the assays and silicon microchip design can be achieved given the benefit of this disclosure, and is expected to lead to shorter cycle and total reaction times. For instance, the HCV assay was able to be performed in 40 cycles with an increase in the length of the RT step time while the other assays needed 50 cycles to maintain sensitivity and efficiency with a shorter RT step. This suggests that cycling time optimization for each target is necessary for sensitive and efficient assays in silicon microreactors. Utilizing these assays on silicon microchip-based diagnostic devices may significantly decrease the time-to-result of molecular POC diagnostic devices.
The flexibility and scalability of silicon microchips allows for integration with other fluidic solutions. For instance, these microreactors may be coupled with microfluidic plasma separation and nucleic acid extraction solutions resulting in a POC diagnostic device that accepts low volume (<50 μL) whole blood specimens without the need of sample preparation other than blood collection. The standard concentration range used in this study was based on concentrations of viral nucleic acids in body fluids during acute and chronic viral infection. Using the standard concentration range in this study and a hypothetical device containing coupled microfluidic plasma separation and nucleic acid extraction, one would be able to detect down to 640 cp/mL plasma assuming a 50 μl, blood specimen (25 μl, of plasma), 50% on-chip recovery of plasma, and 50% on-chip nucleic acid extraction yield. This detection limit would allow for sensitive diagnosis of most acute and chronic viral infections. For example, non-human primate studies have demonstrated peak ZIKV RNA levels in plasma (>105 cp/mL plasma) for the first week of acute infection at levels significantly higher than our theoretical limit of detection. Additionally, this theoretical lower limit of detection would allow for detection of >99.5% of chronic HCV infections. Therefore, incorporation of these ultralow volume reactions in silicon microchip-based molecular diagnostics can result in sufficient sensitivity to diagnose acute and chronic viral infections.
It will be recognized from the foregoing description of this Example that silicon microchips with an etched meandering microreactor, patterned aluminum heaters, resistance temperature detectors, thermal insulation trenches and microfluidic channels for delivery of reagents were produced and precise heating of only the microreactor was demonstrated. Then, the benchscale viral RNA and DNA assays were successfully transferred to the 1.3 μL silicon microreactor. The on-chip reaction sensitivity and efficiency were similar to the bench scale assays but importantly, the 25 min or less reaction time was significantly shorter than the benchscale assays. This disclosure of this Example can be combined with silicon microchip-based nucleic acid extraction methods discussed above, with the intent to shift viral nucleic acid detection and diagnosis from advanced clinical laboratories to the POC.
In conclusion, this Example and those above it provide silicon microchip molecular diagnostic devices for viral infections, and for detecting any polynucleotides. We developed bench scale PCR-based assays for detection of RNA and DNA viruses that were successfully transferred to silicon microchip microreactors with minimal optimization. This finding indicates that the silicon microchip technology can be adapted to detect a broad range of pathogens without the need for significant microchip design or assay optimization. Coupling these with on-chip plasma separation and nucleic acid extraction can provide a platform for the development of rapid and sensitive sample-to-result POC molecular diagnostic solutions that should significantly decrease assay time-to-result, thereby increasing diagnostic certainty in all clinical settings.
Although the present disclosure has been described in connection with preferred embodiments thereof, it will be appreciated by those skilled in the art that additions, deletions, modifications, and substitutions not specifically described may be made without departing from the spirit and scope of the present disclosure as defined in the appended claims.
This application claims priority to U.S. Provisional Application No. 62/464,097 titled “SYSTEM AND METHOD FOR PURIFYING AND AMPLIFYING NUCLEIC ACIDS”, filed Feb. 27, 2017, and U.S. Provisional Application No. 62/554,870 titled “SYSTEM AND METHOD FOR PURIFYING AND AMPLIFYING NUCLEIC ACIDS”, filed Sep. 6, 2017, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/19438 | 2/23/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62464097 | Feb 2017 | US | |
| 62554870 | Sep 2017 | US |
